The proposed research is designed to gain detailed information about the molecular structure of plasma membranes. It is generally accepted that biomembranes are composed primarily of proteins and amphiphilic lipids arranged in biomolecular arrays and that these bilayers will separate into two portions, "halves", upon freezing and fracturing. I have developed a "bulk" splitting method to produce membrane fractions enriched in outer or inner halves, used it to analyze the transmembrane distribution of cholesterol in human erythrocytes (RBC's), and will continue to use it to study other RBC lipids and polypeptides. Methods to produce large flat surfaces of single membrane thickness have been developed using non-vesicular purple membrane fragments isolated from Halobacterium halobium. Membranes can be applied to polylysine-treated glass under conditions that produce the specific attachment of either the cytoplasmic or extracellular surface. These orientation techniques will be used to examine details of the surface chemistry of the bacteriorhodopsin molecule, the transmembrane position of its chromophore, retinal, and the distribution of its lipids. A new electron microscopic method that combines monlayer freeze-fracture (above) with electron microscopic autoradiography (MONOFARG) is currently being developed to examine the in-plane location of radioisotopic molecules as well as the kinetics of their transmembrane diffusion. Finally, a third method, also based on non-random splitting of oriented membranes, that utilizes double-labeling techniques will be developed to examine further kinetic properties. These three techniques can provide information about membrane-associated native molecules or synthetic probes not easily studied by other methods. When developed, the double-labeling approach can provide information about the transmembrane distribution of clinically important molecules with the rapidity appropriate to a diagnostic tool.